生菜紫色调控新基因（PLL5-7）的遗传克隆及生菜叶色的遗传网络解析

Lettuce (Lactuca sativa) is one of the most important vegetables worldwide, and its leaf color is an important agronomic trait. Purple lettuces contain abundant anthocyanins, which provide health benefit to consumers. The purple color in lettuce is controlled by multiple loci, including both qualitative and quantitative loci. In this proposed study, we identified three new loci controlling leaf color in lettuce. BSA in combination with RNA-seq is used to clone corresponding genes. One of them, PLL5, was fine mapped and a candidate gene encodes a WD40-repeat protein that is phylogenetically and structurally related to the RUP1/RUP2 protein in Arabidopsis, which interacts directly with UVR8 as potent repressors of UV-B signaling. We are going to analyze the molecular mechanism of PLL5 involved in UV-B-induced anthocyanin biosynthesis. We have also genetically mapped and cloned the second gene, PLL6, which encoded a MYB transcription factor. We will genetically map and clone the third gene, PLL7, a positive regulator in anthocyanin biosynthesis. This study will provide insights on the genetics controlling leaf color and the mechanism for anthocyanin biosynthesis in lettuce. Above three genes will be combined with the four genes cloned previously to investigate the genetic network underlying the anthocyanin accumulation in lettuce, using the gene expression network as a reference. We will generate lettuce genotypes with diverse intensity of leaf color and diverse types of anthocyanins though various combination of the 7 genes controlling anthocyanin accumulation.

生菜叶色不仅是重要感官品质，产生紫色的花青素还有重要的保健功能。生菜叶色受多位点调控。本项目在前期克隆的4个紫色调控基因基础上，新发现了3个生菜紫色调控位点（PLL5-7）。利用前期成功应用的BSA+RNA-seq遗传研究方法，已经确定紫色调控基因PLL5为UV-B信号传导途径中的负调控基因；PLL6基因编码MYB类转录因子；PLL7定位于1号染色体。本项目将研究PLL5在UV-B诱导的花青素合成途径中的作用；PLL6启动子对基因表达的影响；精细定位并克隆紫色调控基因PLL7，分析其作用机理。本课题还将分析生菜中所有7个叶色控制基因互作关系；通过基因差异表达分析及花青素相关基因的表达网络，挑选10个左右基因进行CRISPR/Cas9敲除，从而深入解析生菜叶色的调控网络。通过杂交创建7个位点不同组合、不同叶色、不同花青素的生菜类型。

生菜叶色不仅是重要感官品质，也是重要的营养品质，紫色的花青素具有重要的保健功能。前期研究表明生菜叶色受多位点调控，本项目通过扫描不同种质资源，构建不同群体，精细定位了紫色生菜中3个控制叶颜色的基因，转基因验证了2个候选基因功能，并系统分析它们的作用机理。PLL5编码一个WD40转录因子，该基因为负调控花青素的合成。在深红亲本中，存在一个氨基酸的改变，导致其功能丧失，使叶片红色加深。PLL6编码MYB转录因子，与PLL2为等位基因。但是PLL6调控生菜叶色的形成是由于启动子区TA重复数量的变化。当TA重复为5次时，生菜叶片表现为全红。而当启动子区TA重复如0次，3次，4次，甚至是8次时，转基因植株叶片只会出现斑点的表型，而不是全红的叶片。生菜中的红色斑也是通过相应的花青素途径，并不存在特异的调控途径。我们推测组蛋白修饰可能参与了生菜红色斑点的形成。我们根据已经克隆和已有的分子标记对加深颜色的基因进行了聚合，挑选PLL1-7位点促进花青素的形成，而ANS基因正常和发生突变的材料，创制了高花青素和高黄酮醇含量的生菜材料。通过本项目的研究克隆了2个生菜中紫色控制基因，明确了它们的作用机理，为一步了解花青素的合成与调控以及培育高含量花青素的生菜品种奠定分子基础。